A recombinant protein of monkey procalcitonin, monoclonal antibody, and preparation method and application thereof

Abstract

The present invention provides a recombinant protein of monkey procalcitonin, a monoclonal antibody thereof, and a preparation method and application thereof. The amino acid sequence of the recombinant protein of monkey procalcitonin is shown as SEQ ID No.1, and the nucleotide sequence encoding the recombinant protein is shown as SEQ ID No.2. By optimizing the antigen peptide sequence and expression vector, the high-efficiency and soluble expression of monkey procalcitonin in Escherichia coli is achieved. A murine monoclonal antibody specifically binding to monkey procalcitonin is screened based on the recombinant protein of monkey procalcitonin, and a pair of monoclonal antibodies that can be paired is screened using the above murine monoclonal antibody. Further, a double-antibody sandwich immuno-ELISA detection method or kit for the recombinant protein of monkey procalcitonin is constructed based on the pair of monoclonal antibody combinations, which has high detection sensitivity and can be well applied to the detection of monkey serum.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a recombinant protein of monkey procalcitonin, a monoclonal antibody, and a preparation method and application thereof. Background Art

[0002] Infectious diseases are extremely common clinically, and laboratory tests are crucial for the diagnosis and treatment of infectious diseases. The four most commonly used indicators are C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), and interleukin-6 (IL-6). The details of the four indicator tests are as follows in the table:

[0003]

[0004] Procalcitonin (PCT) is the precursor of calcitonin (CT), a protein, and its level in plasma increases when there are severe bacterial, fungal, parasitic infections, sepsis, and multiple organ failure (possibly related to bacterial endotoxin). PCT does not increase in cases of autoimmunity, allergy, and viral infection. Localized limited bacterial infections, mild infections, and chronic inflammation do not cause its increase.

[0005] Laboratory animals are the main experimental subjects for preclinical evaluation of drugs, and monkeys are an important species for preclinical research. The application of laboratory animals in new drug research and development covers the preclinical research and clinical research stages, especially more in preclinical research. The pharmacological effects of drugs are evaluated through different experimental methods, their action mechanisms are studied, and their toxic effects are observed to prove the effectiveness and safety of the drugs. Monkeys are the closest to humans in terms of genetic relationship, with 75% - 98.5% homology with human genetic material, making them the animal with the highest homology with humans and thus the ideal animal closest to humans in experiments.

[0006] The amino acid sequence of monkey procalcitonin that can be currently queried is shown as SEQ ID No.11, and the corresponding nucleotide sequence is shown as SEQ ID No.12. However, the antibodies prepared from the protein expressed using this sequence have relatively low sensitivity and cannot meet the application of the monkey procalcitonin ELISA kit. Currently, there is no ELISA detection kit with high sensitivity for monkey procalcitonin applied to the detection of procalcitonin, one of the four major indicators of monkey inflammation. Summary of the Invention

[0007] In view of this, the present invention provides a recombinant protein of monkey procalcitonin, a monoclonal antibody, a preparation method thereof, and its application in ELISA. The present invention uses bioinformatics software to optimize and select a polypeptide of monkey procalcitonin, which is predicted to be expressed in Escherichia coli. Through immunization and screening, a specific paired monoclonal antibody of monkey procalcitonin is obtained, and a double-antibody sandwich ELISA detection system is established, providing a new approach for the study of preclinical monkey inflammation models.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] One object of the present invention is to provide: a recombinant protein of monkey procalcitonin, and the amino acid sequence of the recombinant protein of monkey procalcitonin is shown in SEQ ID No.1.

[0010] Another object of the present invention is to provide: a nucleotide sequence encoding the above recombinant protein of monkey procalcitonin, and the nucleotide sequence is shown in SEQ ID No.2.

[0011] Another object of the present invention is to provide: a preparation method of the above recombinant protein of monkey procalcitonin, comprising the following steps: first, connecting the gene encoding the recombinant protein to an expression vector to construct a recombinant plasmid; then transforming the recombinant plasmid into competent cells, inducing expression and purifying, thereby obtaining the recombinant protein of monkey procalcitonin.

[0012] Furthermore, the gene encoding the recombinant protein is codon-optimized by an E.coli expression system;

[0013] The expression vector is any one of pET28a, pET32a, pET40b, pSmart1, and pCold-TF;

[0014] The competent cells are any one of BL21, Rosetta, and OrigamiB.

[0015] Another object of the present invention is to provide: a monoclonal antibody that specifically reacts with the above recombinant protein of monkey procalcitonin, and the monoclonal antibody includes PCT-5# and PCT-16#;

[0016] Among them, the amino acid sequence of the heavy chain variable region of the monoclonal antibody PCT-5# is shown in SEQ ID No.3, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.4, the amino acid sequence of the light chain variable region is shown in SEQ ID No.5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.6;

[0017] The amino acid sequence of the heavy chain variable region of the monoclonal antibody PCT-16# is shown in SEQ ID No.7, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.8, the amino acid sequence of the light chain variable region is shown in SEQ ID No.9, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.10.

[0018] The fifth object of the present invention is to provide: A method for preparing the above monoclonal antibody, comprising the following steps:

[0019] S1. Add an adjuvant to the above-mentioned recombinant protein of monkey procalcitonin, mix, and immunize female BALB / c mice.

[0020] S2. Take the spleen cells of the immunized mice and fuse them with SP2 / 0 myeloma cells in the logarithmic growth phase, screen and culture to obtain hybridoma cells.

[0021] S3. Expand and culture the obtained hybridoma cells, intraperitoneally inject female BALB / c mice, collect ascites, purify and screen after centrifugation to obtain the monoclonal antibody.

[0022] The sixth object of the present invention is to provide: An ELISA detection method based on the above monoclonal antibody.

[0023] The seventh object of the present invention is to provide: The application of the above ELISA detection method in detecting monkey procalcitonin protein.

[0024] The eighth object of the present invention is to provide: A monkey serum detection kit, which contains the above monoclonal antibody.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) The present invention provides a recombinant protein of monkey procalcitonin, and through optimizing the antigen peptide sequence and expression vector, realizes the high-efficiency, soluble and active expression of monkey procalcitonin in Escherichia coli.

[0027] (2) The present invention screens out a murine monoclonal antibody that specifically binds to monkey procalcitonin based on the recombinant protein of monkey procalcitonin, and uses the above murine monoclonal antibody to screen a pair of paired monoclonal antibodies. Further, a double antibody sandwich ELISA detection method or kit for monkey procalcitonin is constructed based on the combination of this pair of monoclonal antibodies, which can specifically detect monkey procalcitonin protein and has a high detection sensitivity (reaching 13 pg / mL), providing a new approach for preclinical research on monkey inflammation models. Description of the Drawings

[0028] Figure 1 It is the electrophoresis diagram of the recombinant protein of monkey procalcitonin described in Example 1 of the present invention.

[0029] Figure 2 This is the standard curve graph for the ELISA detection of monkey procalcitonin described in Example 3 of the present invention. Detailed implementation manners

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without making creative efforts shall fall within the scope protected by the present invention.

[0031] Example 1

[0032] This example provides a recombinant monkey procalcitonin protein and its preparation method. The amino acid sequence of the recombinant monkey procalcitonin protein is shown as SEQ ID No.1, and the gene sequence encoding the recombinant protein is shown as SEQ ID No.2. The specific sequence information is as follows:

[0033] SEQ ID No.1: MGFQKFSPFLALSILVLLQSGSLHAAPFRDGRESSPDLSEEEARLLLAALVQDYVQMNKFELEQEQETEGSSLDSKRSKRCGNLDACMLGTYTDFNKFATFPQTAIGVGAPMSSDLERNHRRYVSMPQDAN.

[0034] SEQ ID No.2: atgggctttcagaaatttagcccgtttctggcgctgagcattctggtgctgctgcagagcggcagcctgcatgcggcgccgtttcgcgatggccgcgaaagcagcccggatctgagcgaagaagaagcgcgcctgctgctggcggcgctggtgcaggattatgtgcagatgaacaaatttgaactggaacaggaacaggaaaccgaaggcagcagcctggatagcaaacgcagcaaacgctgcggcaacctggatgcgtgcatgctgggcacctataccgattttaacaaatttgcgacctttccgcagaccgcgattggcgtgggcgcgccgatgagcagcgatctggaacgcaaccatcgccgctatgtgagcatgccgcaggatgcgaac.

[0035] Preparation method of recombinant monkey procalcitonin protein, comprising the following steps:

[0036] (1) Plasmid construction: Use computational software to predict and select the one containing the main antigenic epitopes of monkey procalcitonin protein for expression. For example, the sequence shown in SEQ ID No. 2 can be expressed in the E. coli prokaryotic expression system. The gene is cloned into the pET28a vector through the E. coli expression system to obtain the recombinant plasmid pET28a-PCT expressing monkey procalcitonin protein.

[0037] (2) Induced expression: Transform the recombinant plasmid pET28a-PCT into BL21(DE3) competent cells. Spread the transformed bacteria on an LB agar plate containing 50 μg / mL kanamycin and culture overnight at 37 °C. Pick a single colony and inoculate it into 4 mL of LB medium containing 50 μg / mL kanamycin, and culture overnight at 37 °C with shaking at 230 rpm. Inoculate according to 1% of the total volume of the medium into the LB medium containing 50 μg / mL kanamycin, and culture with shaking at 37 °C and 220 rpm for about 3 hours until OD 600 reaches 0.6. Add IPTG with a final concentration of 0.1 mM, and induce at 16 °C and 180 rpm for 15 hours, then collect the bacterial cells.

[0038] (3) Purification of recombinant protein: Since the expressed recombinant protein carries a histidine tag, use a protein purifier and a chromatography column for preliminary crude purification, and then obtain the purified recombinant protein, namely recombinant monkey procalcitonin protein, by ion exchange chromatography. The amino acid sequence is as shown in SEQ ID No. 1.

[0039] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis: Take samples and add the same volume of 2×SDS loading buffer, place in a boiling water bath for 10 min, centrifuge at 12000 rpm for 3 min, take the supernatant and add it to the sample well. After detection, the purified protein is recombinant monkey procalcitonin protein, and the electrophoresis pattern is as Figure 1 shown.

[0040] Example 2

[0041] This example provides a method for screening monoclonal antibodies based on the recombinant monkey procalcitonin protein prepared in Example 1. The specific operation is as follows:

[0042] S1. Mouse immunization: Mix 50 μg of recombinant procalcitonin protein of monkey with an equal volume of Freund's complete adjuvant to obtain a completely emulsified mixture, and subcutaneously inject the mixture into 6-week-old female BALB / c mice at multiple points for immunization. At week 0 and week 2, emulsify an equal proportion of Freund's incomplete adjuvant and recombinant protein according to the above method and immunize again. At week 6, collect mouse serum to detect the antibody titer, and select the mouse with the highest titer to boost immunize 20 μg of recombinant procalcitonin protein of monkey through the tail vein. Three days later, take the mouse spleen for the preparation of hybridoma cells.

[0043] S2. Screening of hybridoma cell lines: After fusing all spleen cells of immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, place them in HAT medium for screening culture. When the fused cells grow to 1 / 2 of the bottom of the well, screen and obtain positive clones by the indirect ELISA method of procalcitonin protein of monkey, and achieve a positive rate of 100% through 2-3 consecutive limited dilution cloning methods, and expand the culture and cryopreservation of the cell lines.

[0044] Among them, the method for screening positive clones by the indirect ELISA method is as follows: First, coat the recombinant protein in a microplate: The coating buffer is carbonate buffer (prepared by dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1 L of pure water), the coating concentration is 1 μg / mL, 50 μL / well, overnight at 4°C; Block with 3% sucrose and 1% BSA, 150 μL per well, 2 hours at 37°C, wash the plate once with washing solution PBST (PBS containing 0.05% Tween-20), and pat dry; Add 50 μL of cell culture supernatant and react at 37°C for 30 min. Discard the liquid in the well, wash the plate 4 times with PBST washing solution, pat dry and add 50 μL of HRP-labeled goat anti-mouse (diluted 1:5000 with PBS) per well, react at 37°C for 30 min, wash the plate 4 times again, pat dry and add 50 μL of TMB chromogenic solution per well for color development at room temperature for 10 min, and finally add 0.5 M sulfuric acid as a terminator to terminate the reaction, and measure the OD 450nm value.

[0045] S3. Preparation of monoclonal antibody ascites: After expanding the culture of the screened monoclonal cell line, inject 0.2 mL (containing 2.5×10 6 cells) of female BALB / c mice pretreated with Freund's incomplete adjuvant into the abdominal cavity. About 10 days later, when the mouse abdomen is significantly swollen, collect ascites using a sterile syringe needle. Centrifuge the collected ascites at 3000 r / min for 10 minutes and collect the middle layer.

[0046] S4. Purification of monoclonal antibody: Centrifuge the ascites at 12,000 r / min for 5 minutes, take 5 mL of the supernatant, add 20 mL of acetate buffer (68 mmol / L, pH 4.5), mix well, then slowly add 50 μL of n-octanoic acid while stirring, continue to stir for 30 minutes after adding, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the supernatant. Filter the supernatant through absorbent cotton, add saturated ammonium sulfate at a ratio of 50% (V / V) of the final volume while stirring, continue to stir for 30 minutes after adding, precipitate at 2 - 8 °C for 4 - 5 hours, centrifuge at 12,000 r / min for 30 minutes at 2 - 8 °C, and take the precipitate. Resuspend the precipitate with 3 mL of Tris-HCl (10 mmol / L, pH 9.0), load it into a dialysis bag (MW: 8000 - 14000), and dialyze in 2 L of Tris-HCl (10 mmol / L, pH 9.0) solution at 2 - 8 °C for 14 hours. Transfer the liquid in the dialysis bag to a centrifuge tube, centrifuge at 12,000 r / min for 5 minutes, and the supernatant is the purified monoclonal antibody. Determine the concentration of the purified monoclonal antibody with an ultra-micro spectrophotometer and store it at -20 °C, avoiding repeated freezing and thawing.

[0047] S5. Identification of the binding activity of monoclonal antibody: Use the indirect ELISA method to coat the recombinant protein of monkey procalcitonin and the control vector protein (recombinant protein of monkey SAA, manufacturer: Abcam, catalog number ab110685) respectively, perform ELISA detection on the purified antibody to identify the affinity of the monoclonal antibody, dilute the antibody to 1 μg / ml, and the results are shown in Table 1. In Table 1, "Ctrl" represents the negative control monkey SAA mouse monoclonal antibody.

[0048] Table 1 Identification results of the binding activity of monoclonal antibody

[0049]

[0050] As can be seen from Table 1, for the 21 strains of monoclonal antibodies against monkey procalcitonin screened, the binding activities of the monoclonal antibodies to monkey procalcitonin protein and the control vector protein were detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey procalcitonin protein.

[0051] Example 3

[0052] This example provides the screening and application of paired monoclonal antibodies, as follows:

[0053] 1. HRP labeling of antibody

[0054] Perform HRP labeling operation on the screened monoclonal antibodies according to the classical sodium periodate method. Finally, dialyze the labeled antibody overnight in a buffer of 0.01 M PBS, pH 7.4, add glycerol in a volume ratio of 1:1, and store it in aliquots at -20 °C.

[0055] 2. Establishment of Double Antibody Sandwich Immunoassay Method

[0056] Purified monoclonal antibodies were respectively diluted to concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate made up to 1 L of pure water, pH 9.6), and 50 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate for overnight coating at 4°C. The next day, the coating solution was discarded, and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA, 150 μL per well, incubated at 37°C for 2 h, the blocking solution was discarded, and patted dry. The sample to be tested, procalcitonin protein of monkey, and the control sample, recombinant SAA protein (manufacturer: Abcam, catalog number ab110685) were diluted with PBS to 2 μg / mL, 1 μg / mL, and 0.5 μg / mL and then added to the ELISA plate, 50 μL per well, incubated at 37°C for 35 min, washed 4 times with PBST washing solution, patted dry, and then added with enzyme-labeled monoclonal antibody diluted 500, 1000, and 2000 times with PBS, 50 μL per well, incubated at 37°C for 35 min, washed 4 times again, patted dry, added with 50 μL of TMB chromogenic solution per well, developed color at room temperature for 10 min, and finally added 50 μL of 0.5 M sulfuric acid per well to terminate the reaction, and the OD 450nm value was measured with an enzyme-linked immunosorbent assay reader.

[0057] Then, by adjusting the dilution ratios of the coating antibody and HRP antibody, the most suitable reaction conditions were determined. Specifically: monoclonal antibody was coated in the microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate made up to 1 L of pure water), the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL per well, overnight at 4°C; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 3% sucrose and 1% BSA, 150 μL per well, incubated at 37°C for 2 h, patted dry and stored dry for later use; the recombinant procalcitonin protein of monkey was diluted with PBS to a concentration of 10 ng / ml, 50 μL was added to the microplate coated with monoclonal antibody, and at the same time, SAA protein was diluted to 10 ng / ml as a negative control, reacted at 37°C for 30 min. The liquid in the wells was flicked out, the plate was washed 4 times with PBST washing solution, patted dry; the HRP-labeled monoclonal antibody was diluted with PBS to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL, 50 μL per well was added, reacted at 37°C for 30 min, washed 4 times again, patted dry, added with 50 μL of TMB chromogenic solution per well, developed color at room temperature for 10 min, and finally added the termination solution to terminate the reaction, and the OD 450nm value was measured with an enzyme-linked immunosorbent assay reader. The coating conditions with the most obvious difference between negative and positive results and the optimal reaction conditions of the HRP-labeled antibody were selected.

[0058] By separately coating the monoclonal antibody and labeling it with HRP as described above, and based on the detection of positive and negative samples, the optimal paired combination was selected as the one with the most obvious difference in the detection values of positive and negative samples. Finally, it was determined that: the coated monoclonal antibody was PCT-5# with a concentration of 1 μg / mL, and the HRP-labeled monoclonal antibody was PCT-16# with a concentration of 1.5 μg / mL.

[0059] The full-length amino acid sequence of the variable region of the heavy chain (H chain) of the monoclonal antibody PCT-5# is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4; the full-length amino acid sequence of the variable region of the light chain (L chain) is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6.

[0060] SEQ ID No.3: ELQLVESGGGLVQPGRSLKLSCVASGTRGGFTSMTWVRQAPGKGLEWIASIYGSGNRTYYPDSVRGRFTISRDDAKSILYLQMNSLRSEDTATYYCVRGSYYFYSNFFDTYGQGVMVTVSS.

[0061] SEQ ID No.4: gaactgcagctggtggaaagcggcggcggcctggtgcagccgggccgcagcctgaaactgagctgcgtggcgagcggcacccgcggcggctttaccagcatgacctgggtgcgccaggcgccgggcaaaggcctggaatggattgcgagcatttatggcagcggcaaccgcacctattatccggatagcgtgcgcggccgctttaccattagccgcgatgatgcgaaaagcattctgtatctgcagatgaacagcctgcgcagcgaagataccgcgacctattattgcgtgcgcggcagctattatttttatagcaacttttttgatacctatggccagggcgtgatggtgaccgtgagcagc.

[0062] SEQ ID No.5: DIQMTQSPSLLSASVGDRVTLNCKASQNYRGNLAWYQQKLGEAPKFLIFNSKSLETGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCQTDRFSTTFGGGTKLELKR.

[0063] SEQ ID No.6: gatattcagatgacccagagcccgagcctgctgagcgcgagcgtgggcgatcgcgtgaccctgaactgcaaagcgagccagaactatcgcggcaacctggcgtggtatcagcagaaactgggcgaagcgccgaaatttctgatttttaacagcaaaagcctggaaaccggcgtgccgagccgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcagccggaagatgtggcgacctatttttgccagaccgatcgctttagcaccacctttggcggcggcaccaaactggaactgaaacgc。

[0064] The full-length amino acid sequence of the variable region of the heavy chain (H chain) of the monoclonal antibody PCT-16# is shown in SEQ ID No.7, and the nucleotide sequence is shown in SEQ ID No.8; the full-length amino acid sequence of the variable region of the light chain (L chain) is shown in SEQ ID No.9, and the nucleotide sequence is shown in SEQ ID No.10.

[0065] SEQ ID No.7: QVTLKESGPGILQPSQTLSLTCSFSGTLDRTYFYGVSWIRQPSGKGLEWLAHITFNGTKRYNPSLKSRLTISKDTSSNQVFLKITSVDTADTATYYCARGSFDRTSYFLVWGAGTTVTVSS。

[0066] SEQ ID No.8: caggtgaccctgaaagaaagcggcccgggcattctgcagccgagccagaccctgagcctgacctgcagctttagcggcaccctggatcgcacctatttttatggcgtgagctggattcgccagccgagcggcaaaggcctggaatggctggcgcatattacctttaacggcaccaaacgctataacccgagcctgaaaagccgcctgaccattagcaaagataccagcagcaaccaggtgtttctgaaaattaccagcgtggataccgcggataccgcgacctattattgcgcgcgcggcagctttgatcgcaccagctattttctggtgtggggcgcgggcaccaccgtgaccgtgagcagc。

[0067] SEQ ID No.9: DIVLTQSPASLAVSLGQRATISCKASQTNSYTRYSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQGGGSPYTFGGGTKLEI。

[0068] SEQ ID No.10: gatattgtgctgacccagagcccggcgagcctggcggtgagcctgggccagcgcgcgaccattagctgcaaagcgagccagaccaacagctatacccgctatagctatatgaactggtatcagcagaaaccgggccagccgccgaaactgctgatttatgcggcgagcaacctggaaagcggcattccggcgcgctttagcggcagcggcagcggcaccgattttaccctgaacattcatccggtggaagaagaagatgcggcgacctattattgccagcagggcggcggcagcccgtatacctttggcggcggcaccaaactggaaatt。

[0069] 3. Linear detection based on the double - antibody sandwich ELISA detection method

[0070] Referring to the aforementioned detection steps, first, the protein of the monkey procalcitonin calibration standard was serially diluted with PBS buffer solution, and the diluted concentrations were 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 63 pg / mL, 31 pg / mL, and 0 pg / mL respectively. Then, 50 μL of the diluted standard protein with different concentrations was taken for detection, and three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is as shown in Figure 2 and, according to the four-parameter fitting result, the linearity reached 0.999.

[0071] 4. Sensitivity detection based on the double-antibody sandwich ELISA detection method

[0072] Use the amino acid sequence of monkey procalcitonin found by query: MGFQKFSPFLALSILVLLQAGSLHAAPFRSALESSPDPATLSEEEARLLLAALVQDYVQMKASELEQEQETEGSSLDSPRSKRCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAPGKKRDMSSDLERNHRRYVSMPQDAN (SEQ ID No. 11); the corresponding nucleotide sequence is atgggctttcagaaatttagcccgtttctggcgctgagcattctggtgctgctgcaggcgggcagcctgcatgcggcgccgtttcgcagcgcgctggaaagcagcccggatccggcgaccctgagcgaagaagaagcgcgcctgctgctggcggcgctggtgcaggattatgtgcagatgaaagcgagcgaactggaacaggaacaggaaaccgaaggcagcagcctggatagcccgcgcagcaaacgctgcggcaacctgagcacctgcatgctgggcacctatacccaggattttaacaaatttcatacctttccgcagaccgcgattggcgtgggcgcgccgggcaaaaaacgcgatatgagcagcgatctggaacgcaaccatcgccgctatgtgagcatgccgcaggatgcgaac (SEQ ID No. 12). Use the protein expressed by this sequence to prepare monoclonal antibodies, and finally prepare a sandwich ELISA. First, gradient dilute the monkey procalcitonin calibration standard protein with PBS buffer solution. The concentrations of the standard products after dilution are 10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312 pg / mL, 156 pg / mL, and 0 pg / mL respectively. Take 50 μL of the diluted standard protein with different concentrations and 20 PBS buffer solutions for detection and perform a sensitivity test. The measured values of the standard curve and the PBS buffer solution are shown in Table 2. According to the formula "average value + 2SD", the sensitivity of the double antibody sandwich ELISA detection method is calculated to be 126 pg / mL.

[0073] Table 2 Sensitivity test data

[0074]

[0075] Referring to the above method, using the technical solution provided by the present invention, first, the monkey procalcitonin calibration standard protein is serially diluted with PBS buffer solution, and the diluted concentrations are 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 63 pg / mL, 31 pg / mL, and 0 pg / mL respectively. Take 50 μL of the diluted standard protein with different concentrations and 20 PBS buffer solutions for detection and perform a sensitivity test. The standard curve and the measured values of the PBS buffer solution are shown in Table 3. According to the formula "average value + 2SD", the sensitivity of the double antibody sandwich ELISA detection method is calculated to be 13 pg / mL.

[0076] Table 3 Sensitivity test data

[0077]

[0078] According to the comparison results, the sensitivity of the sandwich ELISA detection method of the present invention after optimization is significantly higher than that before optimization.

[0079] Example 4

[0080] Furthermore, in order to verify the accuracy of the technical solution provided by the present invention, the following verification examples were also carried out.

[0081] Referring to the aforementioned detection steps, based on the double antibody sandwich ELISA detection method, the OD values of 16 monkey sera without infection and 16 monkey sera with bacterial infection were measured and identified, and the measured OD values are shown in Table 4.

[0082] Table 4 Identification results of non-infected monkey sera and infected monkey sera

[0083]

[0084] It can be seen from the detection results that there are significant differences between the infected and non-infected. This shows that the double antibody sandwich immuno-ELISA detection method provided by this application not only has high sensitivity, but also can accurately detect whether there is an infection in monkey sera.

[0085] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0086] The above are only the preferred implementation schemes of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A recombinant monkey procalcitonin protein, characterized in that: The amino acid sequence of the monkey procalcitonin recombinant protein is shown in SEQ ID No.

1.

2. The nucleotide sequence encoding the monkey procalcitonin recombinant protein according to claim 1, characterized in that: The nucleotide sequence is shown as SEQ ID No.

2.

3. A method for preparing the monkey procalcitonin recombinant protein according to claim 1, characterized in that: The method comprises the following steps: firstly connecting the gene encoding the recombinant protein to an expression vector to construct a recombinant plasmid; then transforming the recombinant plasmid into competent cells, inducing expression and then purifying to obtain the monkey procalcitonin recombinant protein.

4. The preparation method according to claim 3, characterized in that: The gene encoding the recombinant protein is codon-optimized in the E. coli expression system; The expression vector is any one of pET28a, pET32a, pET40b, pSmart1, and pCold-TF; The competent cells are any one of BL21, Rosetta, and OrigamiB.

5. A monoclonal antibody that specifically reacts with the monkey procalcitonin recombinant protein according to claim 1, characterized in that: The monoclonal antibodies include PCT-5# and PCT-16#; The amino acid sequence of the heavy chain variable region of the monoclonal antibody PCT-5# is shown in SEQ ID No.3, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.4, the amino acid sequence of the light chain variable region is shown in SEQ ID No.5, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.6; The amino acid sequence of the heavy chain variable region of the monoclonal antibody PCT-16# is shown in SEQ ID No.7, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.8, the amino acid sequence of the light chain variable region is shown in SEQ ID No.9, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

10.

6. A monkey serum detection kit, characterized in that: The kit contains the monoclonal antibody according to claim 5.

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